CD4 T Cell Immunity Is Critical for the Control of Simian Varicella Virus Infection in a Nonhuman Primate Model of VZV Infection

Primary infection with varicella zoster virus (VZV) results in varicella (more commonly known as chickenpox) after which VZV establishes latency in sensory ganglia. VZV can reactivate to cause herpes zoster (shingles), a debilitating disease that affects one million individuals in the US alone annually. Current vaccines against varicella (Varivax) and herpes zoster (Zostavax) are not 100% efficacious. Specifically, studies have shown that 1 dose of varivax can lead to breakthrough varicella, albeit rarely, in children and a 2-dose regimen is now recommended. Similarly, although Zostavax results in a 50% reduction in HZ cases, a significant number of recipients remain at risk. To design more efficacious vaccines, we need a better understanding of the immune response to VZV. Clinical observations suggest that T cell immunity plays a more critical role in the protection against VZV primary infection and reactivation. However, no studies to date have directly tested this hypothesis due to the scarcity of animal models that recapitulate the immune response to VZV. We have recently shown that SVV infection of rhesus macaques models the hallmarks of primary VZV infection in children. In this study, we used this model to experimentally determine the role of CD4, CD8 and B cell responses in the resolution of primary SVV infection in unvaccinated animals. Data presented in this manuscript show that while CD20 depletion leads to a significant delay and decrease in the antibody response to SVV, loss of B cells does not alter the severity of varicella or the kinetics/magnitude of the T cell response. Loss of CD8 T cells resulted in slightly higher viral loads and prolonged viremia. In contrast, CD4 depletion led to higher viral loads, prolonged viremia and disseminated varicella. CD4 depleted animals also had delayed and reduced antibody and CD8 T cell responses. These results are similar to clinical observations that children with agammaglobulinemia have uncomplicated varicella whereas children with T cell deficiencies are at increased risk of progressive varicella with significant complications. Moreover, our studies indicate that CD4 T cell responses to SVV play a more critical role than antibody or CD8 T cell responses in the control of primary SVV infection and suggest that one potential mechanism for enhancing the efficacy of VZV vaccines is by eliciting robust CD4 T cell responses

Bacterial artificial chromosome derived simian varicella virus is pathogenic in vivo

Abstract Background Varicella zoster virus (VZV) is a neurotropic alphaherpesvirus that infects humans and results in chickenpox and herpes zoster. A number of VZV genes remain functionally uncharacterized and since VZV is an obligate human pathogen, rigorous evaluation of VZV mutants in vivo remains challenging. Simian varicella virus (SVV) is homologous to VZV and SVV infection of rhesus macaques (RM) closely mimics VZV infection of humans. Recently the SVV genome was cloned as a bacterial artificial chromosome (BAC) and BAC-derived SVV displayed similar replication kinetics as wild-type (WT) SVV in vitro. Methods RMs were infected with BAC-derived SVV or WT SVV at 4x105 PFU intrabronchially (N=8, 4 per group, sex and age matched). We collected whole blood (PBMC) and bronchoalveolar lavage (BAL) at various days post-infection (dpi) and sensory ganglia during latent infection (>84 dpi) at necropsy and compared disease progression, viral replication, immune response and the establishment of latency. Results Viral replication kinetics and magnitude in bronchoalveolar lavage cells and whole blood as well as rash severity and duration were similar in RMs infected with SVV BAC or WT SVV. Moreover, SVV-specific B and T cell responses were comparable between BAC and WT-infected animals. Lastly, we measured viral DNA in sensory ganglia from both cohorts of infected RMs during latent infection. Conclusions SVV BAC is as pathogenic and immunogenic as WT SVV in vivo. Thus, the SVV BAC genetic system combined with the rhesus macaque animal model can further our understanding of viral ORFs important for VZV pathogenesis and the development of second-generation vaccines

Summary of effect of immune cell depletions on the anti-SVV response.

<p>Hallmarks of the immune response during acute SVV infection were compared between control and depleted animals. N/A indicates that comparison could not be carried out due to the depletion of the T or B cell subset in question.</p

Representative examples of varicella in control and depleted animals.

<p>Varicella rash on the trunk of a representative (A) control, (B) CD20 depleted, (C) CD8 depleted, and (D) CD4 depleted animal at 10 dpi.</p

Impact of T and B cell depletion on the kinetics and magnitude of T cell proliferation following acute SVV infection.

<p>Frequency of proliferating (Ki67+) CD4 and CD8 T cells within central and effector memory subsets was measured in BAL (A–D) and PBMC (E–H) by FCM. Data points for CD4 T cell Ki67+ frequency in CD4-depleted animals are not shown 7–21 dpi as there were no CD4 T cells in circulation during this time period. Similarly data points for Ki67+ CD8 T cell frequency are not shown 0–14 dpi as there were no CD8 T cells detected during this period. * indicates p<0.05 as compared to control animals.</p

Impact of T and B cell depletion on frequency of SVV-specific T cells.

<p>The frequency of SVV-specific T cells in BAL and PBMCs was measured by intracellular cytokine staining following stimulation with overlapping peptide pools covering ORFs 4, 31, 61 and 63. The average percentage of responding (IFNγ+ and IFNγ+TNFα+) T cells ± SEM within CD4 CM, CD4 EM, CD8 CM and CD8 EM subsets in BAL (A–D) and PBMCs (E–H) is shown. Responses detected on day 0 were on average <0.5% and were subtracted from subsequent time points. * indicates p<0.05 as compared to control animals.</p

Impact of T and B cell depletion on kinetics and magnitude of B cell proliferation and IgG/IgM production following SVV infection.

<p>Frequency of proliferating (Ki67+) B cells within marginal zone-like and memory subsets in BAL (A, B) and PBMC (C, D) was measured using FCM. Data points for B cell proliferation in CD20-depleted animals are not shown 0–14 dpi as there were no B cells in circulation during this time period. Average SVV-specific IgM (E) and IgG (F) end point titers± SEM in control, CD20 depleted, CD8 depleted, and CD4 depleted animals (n = 4/group) were determined by standard ELISA.* indicates p<0.05 as compared to control animals.</p

Efficacy of antibody-mediated depletion of immune cells during acute SVV infection.

<p>(A–C) Average frequency of CD4 T (A), CD8 T (B), and CD20 B (C) cells in bronchial alveolar lavage (BAL) of control, CD20 depleted, CD8 depleted, and CD4 depleted animals (n = 4/group) were measured using flow cytometry (FCM). Absolute numbers per µl/blood were then calculated by converting the percentage of these subsets using complete blood counts obtained at every time point (D–F).</p

Summary of rash duration, lesion number, and disease severity of control and experimental animal groups following acute SVV infection.

<p>The number of lesions for each group was determined by averaging the lesions observed on the abdomen of each animal within each group at 10dpi. The disease severity of each group was determined by averaging the assigned severity score (based on the size and duration of observed vesicles) of each animal within each group (1–2 = mild; 3–4 = moderate; 5 = severe).</p